Nano silicated-FeAl2O4 functionalized by DL-alaninium nitrate ionic liquid (FeAl2O4-SiO2@[DL-Ala][NO3]) as versatile promotor for aqua-mediated synthesis of spiro[chromenopyrazole-indene-triones and spiro[chromenopyrazole-indoline-diones

In this work, the spinel FeAl2O4 was prepared and functionalized step-by-step with silica and alaninium nitrate ionic liquid ([DL-Ala][NO3]) to produce a bio-based multi-layered nanostructure (nano FeAl2O4-SiO2@[DL-Ala][NO3]). The obtained magnetized inorganic-bioorganic nanohybrid characterized by Fourier transform infrared spectroscopy (FT-IR), vibrating-sample magnetometry (VSM), field emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDAX), transmission electron microscopy (TEM), thermogravimetric analysis/differential scanning calorimetry (TGA/DSC), X-ray fluorescence (XRF), and X-Ray diffraction (XRD) analysis. A facile synthesis of some tricyclic dihydro-spiro[chromeno[2,3-c]pyrazole-4,2′-indene]triones and dihydro-spiro[chromeno[2,3-c]pyrazole-4,3′-indoline]diones via domino four-component one-pot reaction of various hydrazine derivatives, ethyl acetoacetate, heterocyclic 1,2-ketones (ninhydrin, isatin, 5-bromoisatin) and cyclic 1,3-diketones (dimedone and 1,3-cyclohexanedine), examined in the presence of nano FeAl2O4-SiO2@[DL-Ala][NO3] nanohybrid in refluxing aqueous media, successfully. The multi-aspect characteristics of the nanohybrid which consist of magnetized inorganic and bioorganic parts, could be the reason of its special catalytic efficacy. The recovery and reusability of the FeAl2O4-SiO2@[DL-Ala][NO3] magnetized nanoparticles (MNPs) were performed in two runs without significant activity loss.

Recently, diverse multi-layered novel magnetized silicated nanostructures get special attention in various fields of science, technology, and catalysis [34][35][36][37] .Several kinds of silicate materials has bene utilized as linkers and/ or supports due to special characteristics such as hydrophilicity (due to their surface silanol groups), biocompatibility, post-functionalization capability, agglomeration avoidance of nano-sized cores, low toxicity, easily surface modification, stability, cost-effectiveness and oxidation preventer of their inner layers 38,39 .
In recent years, several types of magnetized nanohybrids (consist of bio/organic and inorganic parts) get special attention in various fields of science and technology such as nanocarrier for targeted gene delivery into HEK-293 T cells 40 , removal of toxic pollutants from contaminated water 41 , Magnetic resonance/Raman imaging 42 , and catalysts of various transformations 43 .

Characterization of the bio-nanostructure
The preparation procedure and the proposed structure of the magnetized inorganic-bioorganic nanohybrid (nano FeAl 2 O 4 -SiO2@[DL-Ala][NO 3 ]) illustrated in Fig.  3.According to Fig. 3a, the peak at 441 cm −1 , 578 cm −1 related to the stretching vibrations of Fe-O 48 The peak at 1621 cm −1 is attributed to Al-O bond 49 and the peak at and 3438 cm −1 respect to O-H bond.Based on Fig. 3b, the appearance of bands at 1076 cm −1 related to stretching vibration n of Si-O-Si bonds respectively.In addition, broadening the O-H starching band at 3446 cm −1 -3741 cm −1 , which related to Si-OH stretching vibration that combined with O-H groups of FeAl 2 O 4, affirmed embedding of silica on the   FeAl 2 O 4 core.According to Fig. 3c, the peaks at 1529 cm −1 , 1346 cm −1 , and 1140 cm −1 (NO 3 − group) 50 , 1357 cm −1 (bending of methyl group), 1645 cm −1 and 1508 cm −1 (N-H bending vibrations in NH 3 + ), and 1743 cm −1 (C=O stretching vibration), affirmed preparation of the [DL-Ala][NO 3 ] ionic liquid.In addition, appearance of broad peaks in the region of 2763 cm −1 -3317 cm −1 related to the O-H group in CO 2 H, which overlapped by C-H sp 3 and NH 3 + stretching vibrations in [DL-Ala][NO 3 ].Finally, assisting all the mentioned peaks in Fig. 3d, confirmed the synthesis of FeAl 2 O 4 -SiO 2 @[DL-Ala][NO 3 ] bio-nanostructure.The FESEM images of FeAl 2 O 4 -SiO 2 @[DL-Ala][NO 3 ] bio-nanocomposite in 2 µm and 500 nm magnetization demonstrated in Fig. 5.The results affirmed presence of approximately agglomerated particles with semispherical shape with the average size of 55-95 nm.
The result of the energy-dispersive X-ray analysis and element distribution image (EDAX mapping) represented in Fig. 6.The data revealed the presence of Al (2.20 W%), Fe (54.87 W%), Si (4.36 W%), C (7.37 W%), N (1.16 W%) and O (30.06 W%) elements which confirmed the successful preparation of nanocatalyst.The EDX mapping in addition to the conventional executed EDAX elemental mapping (1 µm) confirmed a meaningful and uniform distribution of the element in bio-nanocomposite.
The TEM images (in 100 nm and 50 nm magnifications) and also particle size distribution histogram of the FeAl 2 O 4 -SiO 2 @[DL-Ala][NO 3 ] bio-nanohybrid presented in Fig. 7. Based on the images, nanocatalyst is made up of various layers that the dark parts related to the internal core while the light moieties around it belongs to sell.The histogram dedicated that the particle size is in a narrow distribution which almost are 15-20 nm.

Investigation of the catalytic activity of nano FeAl 2 O 4 -SiO 2 @[DL-Ala][NO 3 ] in the synthesis of some spiro[chromeno[2,3-c]pyrazole-4,2′-indene]triones and spiro[chromeno[2,3-c] pyrazole-4,3′-indoline]diones
Initially, to investigate the catalytic efficacy of bio-nanocomposite, the reaction of hydrazine hydrate (1a) (1 mmol), ethyl acetoacetate (2) (1 mmol), ninhydrin (3a) (1 mmol), and dimedone (4a) (1 mmol) in the presence of FeAl 2 O 4 -SiO 2 @[DL-Ala][NO 3 ] was chosen as a model reaction to optimize the reaction conditions.According to results in Table 2, different experimental parameters were tested to obtain the optimized situation.www.nature.com/scientificreports/ The reaction was carried out under solvent-free conditions in different temperatures (entries 1-3).According to the results, although the solvent-free conditions didn't get satisfactory results, but elevating the temperature yielded better reaction promotion.In order to obey the green chemistry rules, the model reaction examined in aqueous media (entries 4-7).The results affirmed that water seems to be good choice.The reaction was performed at 90 oC in different amounts of catalyst (entries 4 and 5).Surprisingly the lower amount of 0.02 g reveled better results.In the next attempt, the reaction examined under aqueous reflux conditions (entries 6 and 7).The best results obtained in the presence of 0.02 g of the nano FeAl 2 O 4 -SiO 2 @[DL-Ala][NO 3 ] in aqueous reflux conditions (entry 6).Although the main purpose was performing the reaction under aqueous green conditions, but other solvents also examined (entries 8-11) It must be mentioned that all the reactions performed via domino one-pot manner in which an water-mediated mixture of (1a), (2), and FeAl 2 O 4 -SiO 2 @[DL-Ala][NO 3 ] bio-nanocomposite stirred at room temperature within 30 min until appearance of 3-methyl-1H-pyrazol-5(4H)-one intermediate (checked by TLC).Then the substrates (3a) and (4a) added to the mixture and stirred under reflux conditions for the appropriate time mentioned in Table 2.
In order to evaluate the efficacy of the bio-based multi-layered nanostructure (nano FeAl 2 O 4 -SiO 2 @[DL-Ala] [NO 3 ]) to promote the model reaction, the synthesis of (5a) examined under catalyst-free conditions as well as the presence of each layer of the catalyst.All the entries proceeded via a domino manner in which a mixture of hydrazine hydrate (1a) and ethyl acetoacetate (2), in 1:1 molar ratio, in the presence of the mentioned catalyst mixed in water (3 mL) for 30 min.Then equimolar amounts of ninhydrin (3a) and dimedone (4a) added and refluxed for the mentioned times in the Table 3.According to the results summarized in Table 3, the crucial role of the nanostructure is clear.
Based on the optimized reaction conditions, different spiro[chromeno[2,3-c]pyrazole-4,2′-indene]triones and spiro[chromeno[2,3-c]pyrazole-4,3′-indoline]diones prepared successfully via a domino one-pot situations.The results are summarized in Table 4.The reaction of different hydrazines (hydrazine hydrate, phenylhydrazine, 4-nitrophenylhydrazine, and 2,4-dinitrophenyl hydrazine) with ethyl acetoacetate, ninhydrin, and dimedone occurred successfully to obtain the corresponding 5a-c products.The reaction of hydrazines (hydrazine hydrate and phenylhydrazine) with ethyl acetoacetate, ninhydrin, and 1,3-cyclohexanedione occurred successfully to obtain the corresponding (5d-f) products.In order to obtain diverse spiroheterocycles, isatin utilized, as a heterocyclic 1,2-diketone candidate, which resulted to good results (5g-h).In order to vast the method scope, 5-bromoisatin also yielded successful results with different hydrazines and 1,3-diketones (dimedone and 1,3-cyclohexanedione) that gained the products 5i-n successfully.The known compounds characterized though the comparison of their data with the corresponding authentic samples 33 .The 5n are new compound which specified completely by physical and spectral data.Baes on the data of Table 3, it seems utilizing isatin and 5-bromoisatin instead of ninhydrin yielded the corresponding adducts in shorter reaction times, which could be due to more sterically-hindered structure of ninhydrin in comparison to isatins.Although the reaction route is not clear completely, the plausible reaction mechanism for the formation of (5a) illustrated in Fig. 10 according to the previously reported observation 33 .The reaction could proceed via two pathways.In pathway (I), the pyrazolone (B) is produced via the reaction of hydrazine hydrate (1a) with ethyl acetoacetate (2) in presence of nano FeAl 2 O 4 -SiO 2 @[DL-Ala][NO 3 ] in water.Compound (B) undergoes the tautomeric equilibrium with its enol form (C). The Knoevenagel condensation of (C) with ninhydrin (3a) yielded the intermediate (D).The tautomeric form of dimedone (E) performed the 1,4-nucleophilic addition with α,β-unsaturated ketone (D) led to intermediate (F), which underwent intramolecular dehydrative cyclization to form the desired product (5a).On the other hand, the product (5a), also could gained form the pathway (II), in which the enolic tautomer of dimedone (E) done the dehydrative nucleophilic attack to ninhydrin (3a) to obtain (G).The subsequent Michael addition of (G) with intermediate (C) generate (H).Finally, compound (5a) is obtained via the dehydrative cyclization route of compound (H).Totally, the ionic characteristic of the [DL-Ala][NO 3 ] IL in the outer layer of the bio-nanocomposite, in addition to the acidic properties of the FeAl 2 O 4 core and silica middle layer, causes the activation of different functional groups in the substrates/intermediates.Actually, the bio-nanohybrid FeAl 2 O 4 -SiO 2 @[DL-Ala][NO 3 ], possess high catalytic efficacy due to synergic effects of intrinsic promotion characteristics of each of its layers.

Experimental General
All reagents and solvents purchased from the Merck, Aldrich and Alfa-Aesar companies and used without any additional purification.Melting points were measured using an electrothermal 9200 apparatus and reported uncorrected.FT-IR spectra of catalyst and samples were taken using a Bruker FT-IR (tensor 27) spectrometer with potassium bromide pellets.Magnetization properties were evaluated by VSM (MDKB) apparatus.The morphology, shape and size of the nanocatalyst was observed via FESEM (VEGA\\TESCAN-LMU and mira).Element percentages were estimated with Philips-PW2404 XRF spectrometer.TEM image was taken by a CM120 electron microscope.TGA analysis was recorded using a TA Q600 thermogravimetric analyzer.The EDAX data and elemental mapping were analyzed by a VEGA3 TESCAN and MIRA TESCAN apparatus.The XRD pattern of the nanostructure was carried out by a X′ Pert MPD.Homogenization of the particles was done in a Wise clean (power of 90 W) ultrasonic bath.A UNIVERSAL 320 centrifuge apparatus (5000-10,000 rpm) was applied for the preparation of the bionanohybride. 1H NMR and 13 C NMR spectra were performed using a Bruker Advance-III 300 MHz spectrometer in DMSO-d 6 as solvent.The sample mass was identified by Mass device (MS (5973) Agilent Technologies).

Preparation of nano FeAl 2 O 4
Synthesis of nano FeAl 2 O 4 was done by a chemical co-precipitation technique reported previously with some modifications 48 .First, a mixture of Al(NO 3 ) 3 .9H 2 O (0.375 g, 1 mmol) and FeCl 2 .4H 2 O (0.795 g, 4 mmol) dissolved in distilled water (100 mL) under N 2 atmosphere at 80 °C within 30 min.Next, NaOH solution (2 M, 6 mL) was added dropwise into the stirring mixture for 10 min until the pH reached 12 that checked with pH paper.The mixture stirred at 80 °C under N 2 atmosphere for 30 min.The solid was separated using an external magnet, washed with deionized water (3 × mL).The obtained solid dried at 75 °C overnight to get a black powder.

Preparation of FeAl 2 O 4 -SiO 2
The process was accomplished through a modified procedure 46 .In a flask, a mixture of FeAl 2 O 4 nanoparticles (1 g) were dispersed in a mixture of ammonia (25 wt %, 2 mL), distilled water (20 mL), and absolute ethanol (60 mL) sonicated in a sonic-bath for 30 min.In the next step, a solution consists of tetraethyl orthosilicate (TEOS, 0.5 mL) in absolute ethanol (1 mL) was added dropwise into the FeAl 2 O 4 nanoparticle solution under vigorous stirring at room temperature.The mixture was then stirred for 20 h at room temperature.Finally, the products were separated by an external magnet, and washed with absolute ethanol (3 × 5 mL).The solid was dried at 70 °C for 5 h to yield FeAl 2 O 4 -SiO 2 as a black powder.www.nature.com/scientificreports/

Preparation of [DL-Ala][NO 3 ] ionic liquid
The [DL-Ala][NO 3 ] ionic liquid was prepared based on a previously reported procedure 51 with some modifications Typically, aqueous mixture of HNO 3 (1 M, 20 mL) and DL-alanine (1 M, 20 mL) stirred magnetically at 60 °C at 500 rpm for 5 h, followed by oven-drying at 95 °C for 3 h.] ionic liquid (1 g) was dispersed in distilled water (30 mL) and stirred for 24 h at room temperature.Then, the solid were collected by an external magnet and washed by  distilled water (3 × 10 mL).The obtained precipitate dried at room temperature for 2 h and next dried at 75 °C for 7 h to get the multi-layered bio-nanostructure as a black solid.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Table 3 .
Screening the catalyst efficacy for the synthesis of (5a).